PROJECT SUMMARY: Our overarching programmatic goal is to implement rationally-designed therapeutic strategies to improve transplantation rates and reduce allograft loss by reducing or eliminating HLA antibodies (Abs). Patients who have developed HLA Abs as a result of a previous transplant, pregnancy, or blood transfusion may undergo excessive waiting times prior to transplantation because a compatible donor cannot be found. Recent FDA Workshops have been conducted because traditional antihumoral therapies with intravenous immunoglobulin (IVIg) and plasmapheresis do not provide acceptable outcomes for rejection treatment or removal of HLA Abs prior to transplantation (termed desensitization). Our prior studies indicate that bone marrow plasma cells (BMPCs) are the major source of HLA Ab production. BMPCs remain inadequately characterized and exhibit substantial resistance to current therapies in vivo. Several desensitization strategies, e.g., plasmapheresis, steroids, IVIg and rituximab to deplete CD20+ B cells, have been investigated to eradicate Ab-producing PCs, remove preformed anti-HLA Abs or reverse acute Ab-mediated rejection (AMR) with limited success. A plausible explanation for the shortcomings of these strategies is that they do not deplete BMPCs. To achieve our mission and overcome these obstacles, we propose PC targeted therapies as a therapeutic approach that can be enhanced by conducting appropriate basic science studies with subsequent evaluation in preclinical models. In this Project, we will focus on proteasome inhibitor (PI) therapy as the cornerstone of current PC targeted therapies. In Aim 1, we will employ active site-specific inhibitors and covalent probes to quantitate structural and functional adaptations in proteasomes from the BMPCs of HLA-sensitized patients after in vivo PI therapy. Our prior studies indicate that immunoproteasomes, a highly specialized proteasomal variant, are upregulated in BMPCs resistant to standard PI therapy. Importantly, immunoproteasome-specific inhibitors target BMPCs that are resistant to standard PI therapy. In Aim 2, we will employ a novel ex vivo platform that extends the longevity of patient BMPCs through co-culture with BM stromal cell media. We will integrate the results to reveal rationally- designed drug cocktails that synergistically target drug-resistant BMPCs from human patients and that lack broad off-target toxicities. In Aim 3, we will we will deploy cutting-edge genomic and bioinformatic tools to better define the cellular and genetic heterogeneity of BMPCs. Single cell (sc)RNA-Seq transcriptomic profiling will be performed to identify key genomic variations and signatures that define BMPC heterogeneity, reveal rare cell populations and determine the effect of HLA-sensitization and PI therapy on BMPC subsets. Taken together, the results will create tools that can be generalized to improve antihumoral therapies for kidney, heart and liver transplant. Our highly organized, integrated and diverse team works in concert to bring science and medicine together, resulting in basic and clinically-relevant knowledge that can be translated directly to conquer disease.